This invention relates to an improved process for converting a nitrile to the corresponding carboxylic acid by using an enzyme catalyst having nitrilase activity. More particularly, the instant invention converts 2-methylglutaronitrile to the ammonium salt of 4-cyanopentanoic acid in aqueous solution using an enzyme catalyst having an aliphatic nitrilase (EC 3.5.5.7) activity.
A nitrilase enzyme directly converts a nitrile to the corresponding carboxylic acid ammonium salt in aqueous solution without the intermediate formation of an amide. Nitrilases have been identified in a variety of microorganisms, for example, Kobayashi et al. (Tetrahedron 46:5587-5590 (1990); J. Bacteriology, 172:4807-4815 (1990)) have described an aliphatic nitrilase isolated from Rhodococcus rhodochrous K22 that catalyzed the hydrolysis of aliphatic nitrites to their corresponding carboxylic acid ammonium salts. A stereospecific nitrilase of Alcaligenes faecalis 1650 has been used to resolve racemic nitrites in the manufacture of chiral carboxylic acids, and the gene encoding the nitrilase has been cloned and expressed (WO 00/23577). A nitrilase has been isolated from the thermophilic bacterium Bacillus pallidus strain Dac521 that catalyzed the hydrolysis of aliphatic, aromatic and heterocyclic nitrites (Almatawah et al., Extremophiles 3:283-291 (1999)). A nitrilase from Rhodococcus rhodochrous NCIMB 40757 or NCIMB 40833 has been used to convert acrylonitrile to ammonium acrylate (U.S. Pat. No. 5,998,180). A nitrilase from Comamonas testosteroni has been isolated that can convert a range of aliphatic xcex1,xcfx89-dinitriles to either the corresponding xcfx89-cyanocarboxylic acid ammonium salt or dicarboxylic acid diammonium salt (CA 2,103,616; S. Lxc3xa9vy-Schil et al., Gene 161:15-20 (1995)). The regioselective hydrolysis of aliphatic xcex1,xcfx89-dinitriles to the corresponding xcfx89-cyanocarboxylic acid ammonium salts by the nitrilase activity of Acidovorax facilis 72W has also been reported (Gavagan et al., J. Org. Chem., 63:4792-4801 (1998)).
A combination of two enzymes, nitrile hydratase and amidase, can also be used to convert aliphatic nitrites to the corresponding carboxylic acid ammonium salts in aqueous solution. Here the aliphatic nitrile is initially converted to an amide by the nitrile hydratase and then the amide is subsequently converted by the amidase to the corresponding carboxylic acid ammonium salt. A wide variety of bacterial genera are known to possess a diverse spectrum of nitrile hydratase and amidase activities, including Rhodococcus, Pseudomonas, Alcaligenes, Arthrobacter, Bacillus, Bacteridium, Brevibacterium, Corynebacterium, and Micrococcus. Cowan et al. (Extremophiles 2:207-216 (1998)) have recently reviewed both the nitrilase and nitrile hydratase/amidase enzyme systems of nitrile-degrading microorganism.
2-Methylglutaronitrile is one example of an aliphatic xcex1,xcfx89-dinitrile that can be regioselectively converted to a xcfx89-cyanocarboxylic acid ammonium salt (i.e., the ammonium salt of 4-cyanopentanoic acid) using a biocatalyst. The biocatalytic preparation of 4-cyanopentanoic acid has been described previously in U.S. Pat. No. 5,814,508 and its divisionals U.S. Pat. Nos. 5,858,736, 5,908,954, 5,922,589, 5,936,114, 6,077,955, and U.S. Pat. No. 6,066,490. These patents relate to a process in which an aliphatic xcex1,xcfx89-dinitrile is converted to an ammonium salt of an xcfx89-cyanocarboxylic acid in aqueous solution using a catalyst having an aliphatic nitrilase (EC 3.5.5.7) activity, or a combination of nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) activities. When the aliphatic xcex1,xcfx89-dinitrile is also unsymmetrically substituted at the xcfx89-carbon atom, the nitrilase produces the xcfx89-cyanocarboxylic acid ammonium salt resulting from hydrolysis of the xcfx89-nitrile group with greater than 98% regioselectivity. U.S. Pat. No. 5,814,508 specifically discloses a method for converting 2-methylglutaronitrile to 4-cyanopentanoic acid in aqueous solution, where Acidovorax facilis 72W is subjected to a 10-120 minute heat treatment (35-70xc2x0 C.) before use as an enzyme catalyst. This heat treatment was critical to select for the desirable regioselective aliphatic nitrilase (EC 3.5.5.7) activity while destroying an undesirable, non-regioselective nitrile hydratase activity. 4-Cyanopentanoic acid can then serve as substrate in a one-step chemical process for the commercial preparation of 1,5-dimethyl-2-piperidone. 1,5-Dimethyl-2-piperidone has many uses as an industrial solvent, including electronics cleaning, photoresist stripping, industrial degreasing and metal cleaning, resin cleanup, ink formulations, industrial adhesives, and as a reaction solvent for polymers and chemicals.
For commercial-scale applications using a biocatalyst, immobilizing microbial cells has many known economical advantages compared to the use of unimmobilized cells. Some advantages are the capacity to use them repeatedly, their ease of separation, and their use in continuous reactions. Cell inclusion into a polymer matrix permits entrapment of living or metabolically inactive cells while maintaining high diffusion of product and substrate. Examples of typical matrices for immobilization are sodium alginate (Bucke, Methods in Enzymology 135:175-189 (1987)) or carrageenan (Chibata et al., Methods in Enzymology 135:189-198 (1987)). Methods of entrapment are relatively simple, and gel material is non-toxic and low priced.
xe2x80x9cOperationalxe2x80x9d stability of immobilized cells can be further increased by subsequent treatment of the cell beads with crosslinking agents that covalently crosslink cells with multifunctional reagents, such as glutaraldehyde and polyethylenimine or hexamethylenediamine. In one example, stability was studied with respect to immobilized E. coli cells in kappa-carrageenan for the production of L-aspartic acid (Chibata, I. In Immobilized Microbial Cells; Venkatsubramanian, K., Ed.; ACS Symposium Series 106; American Chemical Society; Washington, D.C., 1979, pp 187-201). With optimized concentrations of hexamethylenediamine and glutaraldehyde used as a crosslinking treatment, the half-life of immobilized cells was remarkably extended to over five times that of untreated immobilized cells.
Furthermore, Birnbaum et al. (Biotechnol. Lett. 3:393-400 (1981)) disclose methods of increasing the physical stability of calcium alginate immobilized cells. One stabilization method uses polyethylenimine treatment (24 hours), followed by glutaraldehyde cross-linking (1-5 minutes). Bead stability was examined by incubating the immobilized cells in 0.1 M sodium phosphate buffer for ten days. Little cell release was noted from the immobilized cells, thereby demonstrating improved bead integrity. At the same time, overall catalyst activity was detrimentally affected by this protocol. Birnbaum suggests this effect was likely due to glutaraldehyde toxicity.
Finally, the preferred order of adding polyethylenimine and glutaraldehyde for directly immobilizing whole microbial cells or microbial cell material has been understood to depend on the sensitivity of the immobilized enzyme activity to glutaraldehyde. U.S. Pat. No. 4,288,552 discloses that glutaraldehyde-sensitive enzymes (such as thiol-enzymes and others with an SH group in or very near the active site of the enzyme molecule) are inactivated by thiol-reactive agents such as glutaraldehyde. For these types of enzyme catalysts, the invention requires that the microbial cell material be treated with polyethylenimine first, the glutaraldehyde being added simultaneously or subsequently, to negate potential loss of enzyme activity. In contrast, U.S. Pat. No. 4,355,105 teaches that it is desirable to introduce glutaraldehyde before polyethylenimine when immobilizing microorganisms whose enzymes are not sensitive to glutaraldehyde. In this instance the resulting immobilized cells are more readily recovered from the aqueous medium than cells immobilized with polyethylenimine pretreatment before glutaraldehyde addition. In neither case are microbial cells entrapped in a gel or polymer matrix before treatment with polyethylenimine and glutaraldehyde.
The problem to solved, therefore, is developing an economical method for producing immobilized cell catalyst having high specific activity and a prolonged period of physical integrity when used as catalyst for converting a nitrile to the corresponding carboxylic acid ammonium salt. More specifically, the art would be advanced by a process using an enzyme catalyst that results in higher yields and higher concentrations of 4-cyanopentanoic acid, and is also more convenient to use than those previously disclosed.
Applicants"" invention is a method for producing a carboxylic acid comprising: a) immobilizing in alginate an enzyme catalyst characterized by a nitrilase activity; b) adding, to the immobilized enzyme catalyst of step a), a first stabilizer and then a second stabilizer, each in an amount and for a time sufficient to crosslink the immmobilized enzyme catalyst, the first stabilizer and the second stabilizer each selected from the group consisting of glutaraldehyde and polyethylenimine, provided the second stabilizer is other than the first stabilizer; c) contacting the product of step b) with a nitrile in a suitable aqueous reaction mixture; d) isolating the carboxylic acid produced in step c) in the form of a salt or an acid; and e) optionally repeating steps c) and d) at least one time. Preferred embodiments of the invention include where the nitrile of step c) is 2-methylglutaronitrile; the aqueous reaction mixture of step c) has a NH4+:Ca2+ ratio greater than 20:1 (more preferably greater than 200:1, and most preferably greater than 750:1) during the course of the reaction; and the carboxylic acid isolated in step d) is 4-cyanopentanoic acid.
The preferred enzyme catalysts are Acidovorax facilis 72W (ATCC 55746), Acidovorax facilis 72-PF-15 (ATCC 55747), Acidovorax facilis 72-PF-17 (ATCC 55745), Escherichia coli SS1001 (ATCC PTA-1177), or Escherichia coli SW9 (ATCC PTA-1175), and the enzyme catalyst are in the form of whole cells or permeabilized microbial cells. When the enzyme catalyst is Acidovorax facilis 72W (ATCC 55746) there is no need to inactivate the nitrile hydratase activity of the enzyme catalyst by a heat treatment before its immobilization in alginate.
Applicants have made the following biological deposits under the terms of the Budapest Treaty:
As used herein, xe2x80x9cATCCxe2x80x9d refers to the American Type Culture Collection International Depository located 10801 University Blvd., Manassas, Va. 20110-1109, U.S.A. The xe2x80x9cATCC No.xe2x80x9d is the accession number to cultures on deposit with the ATCC. The listed deposits will be maintained in the indicated international depository for at least thirty (30) years and will be made available to the public upon grant of a patent disclosing it. The availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action.
The instant invention relates to an improved process for converting a nitrile to the corresponding carboxylic acid ammonium salt using an enzyme catalyst having nitrilase activity. More particularly, the instant invention converts 2-methylglutaronitrile to the ammonium salt of 4-cyanopentanoic acid in aqueous solution using an enzyme catalyst having an aliphatic nitrilase (EC 3.5.5.7) activity. Applicants now disclose that it is possible to immobilize an enzyme catalyst having nitrilase activity in calcium alginate, and, after crosslinking with glutaraldehyde and polyethylenimine, use the resulting catalyst to produce 4-cyanopentanoic acid ammonium salt at concentrations of at least 1.50 M under conditions which were expected to result in the rapid loss of physical integrity of the crosslinked immobilized enzyme catalyst. It was unexpected that a glutaraldehyde treatment of the immobilized cells prior to the addition of PEI could be performed without any measurable loss of nitrilase activity, or that operating the reaction at a ratio of ammonium/calcium ion of at least 750:1 would not affect the physical integrity of the catalyst beads when it has been recommended not to exceed a ratio of 20:1. The enzyme catalyst was reused in many consecutive recycle reactions with no loss of physical integrity.
Applicants additionally disclose that (in contrast to the corresponding enzyme catalyst prepared using carrageenan) the nitrilase activity of glutaraldehyde/polyethylenimine-crosslinked alginate-immobilized enzyme catalyst increases with increasing concentration of dry cell weight in the catalyst beads. The use of the alginate-immobilized enzyme catalyst is advantageous in that it increases the overall product yield based on catalyst usage when compared to a carrageenan-immobilized enzyme catalyst.
Applicants additionally disclose that in the case of immobilizing the enzyme catalyst Acidovorax facilis 72W, the heat-treatment normally required to inactivate an undesirable nitrile hydratase activity was not required when the cells were immobilized in alginate and the resulting enzyme catalyst crosslinked with the stabilizers glutaraldehyde and polyethylenimine. It was unexpected that the immobilization procedure would selectively and completely inactivate the undesirable nitrile hydratase activity, without producing a measurable loss of nitrilase activity, particularly when it is known that the nitrilase can be inactivated by glutaraldehyde.
The process improvements disclosed herein, when compared to previously known methods, result in higher yields of the product (based on weight of product per weight of catalyst) at higher concentrations than previously obtained. The product of the claimed process is useful as a precursor for polymers, solvents (e.g., 1,5-dimethyl-2-piperidone), and chemicals of high value in the agricultural and pharmaceutical industries.
In the application, unless specifically stated otherwise, the following abbreviations and definitions apply:
xe2x80x9cGlutaraldehydexe2x80x9d is abbreviated GA.
xe2x80x9cPolyethyleniminexe2x80x9d is abbreviated PEI.
xe2x80x9c2-Methylglutaronitrilexe2x80x9d is abbreviated 2-MGN.
xe2x80x9c4-Cyanopentanoic acidxe2x80x9d is abbreviated 4-CPA.
xe2x80x9cEnzyme catalystxe2x80x9d refers to a catalyst that is characterized by a nitrilase activity. The catalyst may be in the form of a whole microbial cell or permeabilized microbial cell(s).
xe2x80x9cAqueous reaction mixturexe2x80x9d is used to refer to an aqueous mixture containing water, a calcium salt at a concentration of at least 2 mM, and optionally, a buffer capable of maintaining the initial pH of the reaction between 5 and 10, preferably between 6 and 8.
xe2x80x9cAqueous product mixturexe2x80x9d is used to refer to an aqueous mixture containing a product resulting from the corresponding process step.
Significant improvements to the process for conversion of 2-methylglutaronitrile (2-MGN) to 4-cyanopentanoic acid (4-CPA) ammonium salt are now disclosed. Specifically, the significant improvements of the instant invention result from a process having the following steps: 1) immobilizing an enzyme catalyst characterized by a nitrilase activity, preferably derived from Acidovorax facilis 72W, in alginate, 2) sequentially adding glutaraldehyde and polyethylenimine (preferably in that order) to the immobilized enzyme catalyst in suitable amounts and for times sufficient to effect each stabilizers"" crosslinking, 3) contacting 2-MGN with the crosslinked immobilized enzyme catalyst in a suitable aqueous reaction mixture, 4) isolating 4-CPA as the acid or an ammonium salt, and 5) then recycling the crosslinked immobilized enzyme catalyst at least one time. The advantages of the instant invention can also be realized when using other enzyme catalysts characterized by nitrilase activity (for example, Rhodococcus rhodochrous, Alcaligenes faecalis, Bacillus pallidus, Comamonas testosteroni, Nocardia sp., Acinetobacter sp., and Arthrobacter sp.), for converting a nitrile to the corresponding carboxylic acid ammonium salt.
Enzyme Catalyst Immobilization
Methods for immobilizing the enzyme catalyst were developed using two different polymer matrices, sodium alginate and kappa-carrageenan. Enzyme catalysts in the form of whole cells or permeabilized microbial cells can be immobilized in alginate or carrageenan; methods for permeabilization are well-known to those skilled in the art, and include, but are not limited to, freeze-thaw, or treatment with organic solvents or detergents (Felix, Bioprocess. Technol. 11:259-278 (1991); Felix, Anal. Biochem. 120:211-234 (1982)). Cell immobilization in alginate is advantageous, since the immobilization can be done at temperatures as low as 5xc2x0 C. In contrast, carrageenan immobilization typically requires temperatures of 45-50xc2x0 C., which can result in inactivation of the nitrilase.
Glutaraldehyde (GA) and polyethylenimine (PEI) treatments were added to the immobilization protocols to further increase the mechnical stability of the beads, such that they would be amenable to a high number of consecutive reactions with catalyst recycle. The amount of GA and PEI added to crosslink the alginate bead enzyme catalysts each ranged from 25 wt % per dry cell weight present in the catalyst beads, to 2 wt % per dry cell weight present in the catalyst beads, where the ratio of GA to PEI added to the catalyst beads also ranged from 3:1 to 1:3. The two crosslinking stabilizers are added to the immobilized enzyme catalysts in either order: GA first, then PEI, or PEI first, then GA. The preferred order of adding the two-crosslinking stabilizers was GA first, followed by PEI. The addition of the second stabilizer is delayed for a time sufficient to permit crosslinking by the first stabilizer. The time for GA-crosslinking of the immobilized enzyme catalysts ranged from 5 min to 2 h, preferably 30 min to 1 h. The time for PEI crosslinking of the immobilized enzyme catalysts ranged from 30 min to 24 h, preferably 1 h to 12 h.
It was unknown and unexpected that a combination of GA/PEI would successfully stabilize the immobilized cell catalysts without damage to the specific nitrilase activity, since it was known that glutaraldehyde inactivates the Acidovorax facilis 72W nitrilase enzyme (Example 1). In Cowan et al., (Extremophiles 2:207-216 (1998)) it is disclosed that nitrilase enzymes function by the nucleophilic attack on the nitrile carbon by an activated thiol residue; U.S. Pat. No. 4,288,552 notes that glutaraldehyde-sensitive enzymes, such as thiol-enzymes, are inactivated by glutaraldehyde.
It has been reported that calcium-crosslinked alginate is not stable in the presence of high concentrations of other cations. Specifically, it is not recommended to exceed a ratio of ammonium/calcium ion of 20:1 or 25:1 (Klein, J. and Vorlop, K. D., In Biotechnology Focus I; Finn, R. F., Ed.; Oxford University Press: New York; 1998, pp 325-336; Smidsrod et al., Trends Biotechnol. 8, 71-78 (1990)). Klein et al. states that the ratio of electrolytes (molar sum of Na+, K+, NH4+, Mg2+) to Ca2+ should not be higher than 20:1 for alginates with a high proportion of L-guluronic acid. In the instant application, after crosslinking with glutaraldehyde and polyethylenimine, the alginate gel enzyme catalysts (which contain a high proportion of L-guluronic acid), retain their physical integrity when recycled in at least 195 consecutive batch reactions to produce high concentrations of 4-cyanopentanoic acid ammonium salt. In these reactions, which contain a minimum calcium ion concentration of 2 mM, the ratio of ammonium ion to calcium ion is typically at least 750:1 (see Example 4). Reactions have been run where the ratio of ammonium ion to calcium ion was 200:1, 750:1, and 950:1. These results certainly could not have been predicted.
When running commercial processes, it is desirable to have high specific activities, here defined as enzyme activity/weight of catalyst. In the instant application, nitrilase-specific activity increased with increasing concentration of dry cell weight in alginate beads, but not in carrageenan beads. With carrageenan beads, there was either a small increase of activity (7%) with a 50% increase in dry cell weight at 30xc2x0 C., or a slight decrease in specific activity at 35xc2x0 C. (see Example 4).
Immobilization of Acidovorax facilis 72W (ATCC 55746) in Glutaraldehyde/Polyethylenimine-crosslinked Alginate
The microbe Acidovorax facilis 72W (ATCC 55746) has been previously isolated from soil samples exposed to aliphatic nitriles or dinitriles (U.S. Pat. No. 5,814,508 and its divisionals U.S. Pat. Nos. 5,858,736, 5,908,954, 5,922,589, 5,936,114, 6,077,955, and U.S. Pat. No. 6,066,490). (U.S. Pat. No. 5,814,508 is hereby incorporated by reference.) When Acidovorax facilis 72W is used as a microbial whole-cell catalyst for the hydrolysis of unsymmetrically substituted xcex1-alkyl-xcex1,xcfx89-dinitriles, the corresponding dicarboxylic acid monoamides and dicarboxylic acids are produced in addition to the desired xcfx89-cyanocarboxylic acid. An undesirable non-regioselective nitrile hydratase activity of this whole-cell catalyst produced the undesirable dicarboxylic acid monoamides, which were further converted by an amidase to the corresponding dicarboxylic acid. Enzyme catalysts such as Acidovorax facilis 72-PF-15, Acidovorax facilis 72-PF-17, Escherichia coli SS1001 and Escherichia coli SW91, which are characterized by the nitrilase activity of Acidovorax facilis 72W but do not exhibit the nitrile hydratase activity of Acidovorax facilis 72W, do not produce the undesirable dicarboxylic acid monoamide and dicarboxylic acid byproducts.
Heating a suspension of Acidovorax facilis 72W (ATCC 55746) in a suitable buffer at 35-70xc2x0 C. for a short period of time was found to deactivate the undesirable nitrile hydratase activity without affecting the desirable nitrilase activity. Thus, previous processes for hydrolysis of 2-methylglutaronitrile (2-MGN) to 4-cyanopentanoic acid (4-CPA) ammonium salt with extremely high regioselectivity required that suspensions of Acidovorax facilis 72W be heat-treated at 35-70xc2x0 C. to inactivate unwanted nitrile hydratase activity and eliminate the production of the unwanted byproduct 2-methylglutaric acid (Gavagan et al., Appl. Microbiol. Biotechnol., 52:654-659 (1999); U.S. Pat. No. 5,814,508). This heat-treatment did not produce any loss of nitrilase activity, and was also routinely used in preparing immobilized cell catalysts.
This invention for the commercial production of 4-cyanopentanoic acid provides the further benefit that immobilizing of non-heat-treated Acidovorax facilis 72W cells in alginate removes approximately 90% of unwanted nitrile hydratase activity. Subsequent glutaraldehyde (GA) or glutaraldehyde and polyethylenimine (GA/PEI) crosslinking of alginate-immobilized Acidovorax facilis 72W cells further reduces the rate of production of undesirable byproducts (2-methylglutaric acid) by the catalyst to that observed with cells that were heat-treated and had no detectable nitrile hydratase activity (see Example 10). Applicants"" disclosure is surprising and unexpected, since the heat treatment step heretofore required in U.S. Pat. No. 5,814,508 is no longer needed. It was unknown that the immobilization of Acidovorax facilis 72W cells in alginate, followed by GA or GA/PEI-crosslinking, would selectively and completely inactivate an undesirable nitrile hydratase activity, while at the same time causing no measurable loss in the desirable nitrilase activity. This result is particularly unexpected as it was known that nitrilase is inactivated by glutaraldehyde.
Hydrolysis of 2-Methylglutaronitrile
The temperature of the hydrolysis reaction is chosen to optimize both the reaction rate and the stability of the enzyme catalyst activity. The temperature of the reaction may range from just above the freezing point of the suspension (ca. 0xc2x0 C.) to 60xc2x0 C., with a preferred range of reaction temperature of from 5xc2x0 C. to 35xc2x0 C. The immobilized enzyme catalyst suspension may be prepared by suspending the catalyst in distilled water, or in a aqueous solution of a buffer which will maintain the initial pH of the reaction between 5.0 and 10.0, preferably between 6.0 and 8.0. As the reaction proceeds, the pH of the reaction mixture may change due to the formation of an ammonium salt of the carboxylic acid from the corresponding nitrile functionality of the dinitrile. The reaction can be run to complete conversion of dinitrile with no pH control, or a suitable acid or base can be added over the course of the reaction to maintain the desired pH. A calcium salt, including but not limited to calcium chloride or calcium acetate, is added to the hydrolysis reaction at a concentration of at least 2 mM to maintain the physical integrity of the crosslinked immobilized enzyme catalyst.
4-Cyanopentanoic acid may also be isolated from the product mixture (after removal of the catalyst) by adjusting the pH of the reaction mixture to between 2.0 and 2.5 with concentrated HCl, saturating the resulting solution with sodium chloride, and extracting 4-cyanopentanoic acid with a suitable organic solvent (such as ethyl acetate, ethyl ether, or dichloromethane). The organic extracts are then combined, stirred with a suitable drying agent (e.g., magnesium sulfate), filtered, and the solvent removed (e.g., by rotary evaporation) to produce the desired product in high yield and in high purity (typically 98-99% pure). If desired, the product can be further purified by recrystallization or distillation. Alternatively, the 4-cyanopentanoic acid ammonium salt may be used directly in a subsequent reaction, as is the case in the preparation of 1,5-dimethyl-2-piperidone (U.S. Pat. No. 5,814,508 and its divisionals U.S. Pat. Nos. 5,858,736, 5,908,954, 5,922,589, 5,936,114, 6,077,955 and U.S. Pat. No. 6,066,490).